The TCR alpha-chain undergoes phenotypic allelic exclusion during thymocyte development. This is regulated post translationally and starts to manifest itself concurrent with positive selection. In immature thymocytes, cells with two cell-surface alpha-chains are relatively frequent. We will determine how post-translational allelic exclusion is regulated testing several models, including the "selective retention" model whereby the stimulated TCR alpha-Beta combination is internalized on the cell surface and the unstimulated alpha beta combination is internalized. Stimulation of thymocytes with ant alpha-chain reagents does not result in modulation of TCR from the surface. In contrast, similar stimulation with anti-chain or CD3 regents does result in modulation. How the signaling through alpha-chain differs from that mediate through other parts of TCR will be determined. Transgenic mice will be made with two alpha-chains that can be followed individually, after stimulation by ligands that can cause negative or positive selection through one alpha-Beta combination he effect on the non-stimulated alpha-beta combination will be investigated. How the two alpha-chains differ in recruitment he interface with an APC or bead-bound anti-TCR antibodies will be determined by microscopy. Recruitment of signaling molecules to this region by different anti-TCR or antigenic stimuli will be investigated. Fluorescent construct alpha-chains will be prepared and expressed in cell lines, so that their recruitment and internalization in response different stimuli can be assessed in real time. Transgenic mice with such fluorescent constructs will allow us to investigate allelic exclusion and down-modulation in response to selecting signals. Both c-Cbl and SLAP/Fyb are required for alpha-chain allelic exclusion. C-Cbl is an ubiquitin ligase and targets receptor tyrosine kinases for degradation after signaling. SLAP is involved in linking the activated TCR to the cytoskeleton, and thence to endosomes. The role of these proteins will be determined using knockout mice bred to appropriate TCR transgenic systems. Recruitment of these adapters to the immune synapse will be investigated in double TCR alpha-chain expressing cells. Ubiquitination Cb1 will be investigated under these differing conditions. Fluorescent chimeras of c-Cbl and SLAP will be used in concert with the labeled alpha-chains to be able to measure the recruitment and colocalization in real time. The different effects of stimulation of thymocytes through alpha-chain versus other parts of the TCR will be investigated using DNA array technology. Expression of fluorescent versions of TCR alpha-chain in cell lines and transgenics will allow to investigation of whether the TCR:CD3 unit on the cell surface is monovalent or divalent. The valency of TCR and its ability to dimerize during activation will be tested in cells at differing stages of development and activation.